Liposome complexes for increased systemic delivery

ABSTRACT

Highly efficient cationic liposomes have been developed as an improved delivery system for biologically-active reagents. A novel structure, the sandwich liposome, is formed and comprises one or more biologically active agents internalized between two bilomellar liposomes. This structure protects the incoming agent and accounts for the high efficiency of in vivo delivery and for the broad tissue distribution of the sandwich liposome complexes. 
     These novel liposomes are also highly efficient carriers of nucleic acids. By using extruded DOTAP:cholesterol liposomes to form complexes with DNA encoding specific proteins, expression has been improved dramatically. Highest expression was achieved in the lung, while increased expression was detected in several organs and tissues.

This is a continuation of application Ser. No 09/242,190, filed Oct. 4,1999, now U.S. Pat. No. 6,413,544, which was a 35 U.S.C. 371 filing ofPCT/US97/13599, filed Aug. 1, 1997, which claims priority to provisionalapplication No. 60/024,931, filed Aug. 30, 1996, now abandoned.

FIELD OF THE INVENTION

The present invention is directed to a liposomal preparation which isbased on a composition of specific lipids which form liposomes. It isalso an object of the present invention to provide a method forpreparing a liposomal composition carrying a biologically active agent.The liposomal delivery system of the present invention is used as highlyefficient transfer and therapy methods.

BACKGROUND OF THE INVENTION

Lipidic particles may be complexed with virtually any biologicalmaterial. This capability allows these complexes to be useful asdelivery systems for proteins, therapeutic agents, chemotherapeuticagents and nucleic acids. Although lipidic complexes have been used fora myriad of drug therapies, one area where these delivery systems haveshown promising results is in gene therapy. For gene therapy to besuccessful efficient and safe transfer of genes or biologically activereagent to a target cell is required. Hence the need for improveddelivery systems, in both conventional and gene-based therapies isalways at the forefront.

Lipidic particles have been shown to be efficient vehicles for many invitro and in vivo applications. Lipidic particles complexed with DNAhave been used in vitro (Felgner et al. (1987); Gao et al. (1991)) andin vivo (Nabel et al. (1990); Wang et al. (1987); Zhu et al. (1993);Soriano et al. (1983)) for the expression of a given gene through theuse of plasmid vectors.

Formation of complexes of DNA with cationic lipidic particles hasrecently been the focus of research of many laboratories. Improvedformulations of cationic lipids have greatly increased the efficiency ofDNA delivery to cells in tissue culture (Felgner et al. (1987)). Incontrast, intravenous DNA delivery in animals using cationic liposomeshas been less efficient (Zhu et al. (1993); Philip et al. (1993);Solodin et al. (1995); Liu et al. (1995); Thierry et al. (1995);Tsukamoto et al. (1995); Aksentijevich et al. (1996)) limiting thetherapeutic application of nonviral vectors to gene therapy. Improvedliposome formulations for in vivo delivery is a valuable alternative togene therapy using viral vectors and avoids several problems associatedwith viral delivery. Although efforts to synthesize new cationic lipidsled to the discovery of more efficient transfection agents, theirefficiency measured in tissue culture does not correlate with ability todeliver DNA after systemic administration in animals (Solodin et al.(1995)). Functional properties defined using in vitro experiments do notassess stability of the complexes in plasma or their pharmacokineticsand biodistribution, all of which are essential for in vivo activity(Felgner et al. (1994)). Colloidal properties of the complexes inaddition to the physicochemical properties of their component lipids maydetermine these parameters.

The liposome provides an alternative to viral delivery systems in genetherapy which may involve the transfer of normal, functional geneticmaterial into cells to correct an abnormality due to a defective ordeficient gene product. Typically, the genetic material to betransferred should at least contain the gene to be transferred togetherwith a promoter to control the expression of the new gene.

Methods for viral DNA delivery systems suffer from many inherentproblems including immune responses, inability to deliver viral DNAvectors repeatedly, difficulty in generating high viral titers, and thepossibility of infectious virus. Non-viral delivery methods provide analternative system that is devoid of these problems. However, until now,low efficiency of DNA delivery by liposomes has limited the therapeuticapplication of this technology for gene therapy. The liposomes of thepresent invention have increased systemic DNA delivery and geneexpression up to 150-fold over that previously reported.

Therefore, an object of the present invention is the synthesis of ahighly efficacious liposome structure capable of delivering biologicallyactive agents into a subject.

Another object of the present invention is the use of these stableliposomes as carriers of nucleic acids for delivery and expression ofthe nucleic acid product at a target site within an animal.

Yet another object of the present invention is the use of these stableliposomes as carriers of nucleic acids for delivery and expression ofthe nucleic acid product systemically to a patient.

A further object of the present invention is the production of kitscontaining the stable liposomes of the present invention, capable ofcarrying any nucleic acid of interest.

Another object of the present invention is to use liposomes carryingspecific reagents for human gene therapy in treatment of disease.

Yet a further object of the present invention relates to providing amethod for long-term expression of a gene product from a non-integratednucleic acid in a patient.

SUMMARY OF THE INVENTION

The present invention relates to a novel liposome structure capable ofcarrying biologically-active reagents. These liposome structures arehighly efficient vehicles for delivery of biologically active agents totarget locations in a patient as well as provide systemic reagentdelivery. The liposome complexes of the present invention are small insize and have a net +/− charge (“ρ”) of about 2.

The present invention further relates to a method of preparing thesenovel liposomes comprising the steps of heating, sonicating, andextrusion of the liposome structures. The method of preparation of thepresent invention produces complexes of appropriate and uniform size,which are structurally stable and produce maximal extrusion. Liposomesprepared by this method are also encompassed by the present invention.

The present invention further relates to a novel liposome structurecapable of carrying nucleic acids. The present invention also relates toan improved liposome formulation comprising DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)-propane) and cholesterol(“Chol”) and a nucleic acid which produces exceptionally high geneexpression and protein production in vivo. These formulations areextremely stable, homogeneous in size, and can complex nucleic acidsover a wide range of nucleic acid:liposome ratios. The present inventiondemonstrates up to 150-fold greater gene expression following in vivosystemic delivery in animals as compared to formulations previouslydescribed in the literature.

The present invention also relates to liposomes carrying non-immunogenictargeting ligands and stealth lipids. These ligands facilitate thetargeted delivery of the liposomes to a particular tissue or site in thebody.

The present invention relates to kits containing the present liposomestructure capable of carrying a reagent within it. One such kit maycomprise the liposome structures ready for the user to add thebiological reagent of interest. A kit may further comprise a liposomepreparation and one or more specific biologically-active reagents foraddition to the liposome structure. Another kit of the present inventioncomprises a set of liposome structures, each containing a specific,biologically-active reagent, which when administered together orsequentially, are particularly suited for the treatment of a particulardisease or condition.

The present invention provides a therapeutic method of treatingdiseases, ailments and conditions based upon a liposome-facilitateddelivery of biologically active agents. For example, the presentinvention provides a pharmaceutical liposomal formulation for thedelivery of nucleic acids using systemic administration to providelong-term expression of a given nucleic acid. In addition, the presentinvention encompasses in vitro cell transfection followed by tissuetransplantation such that the transfected cells may be incorporated intransplanted tissue. This method is referred to as in vitro/ex vivotransfer. Other biologically-active agents may be encapsulated in theliposomes of the present invention for in vitro/ex vivo methods so longas a +/− charge of positive 2 is maintained.

The present invention further provides an effective vaccine vehiclecapable of effective delivery, boosting antigen-immune response andlowering unwanted extraneous immune response, presently experienced withadjuvants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Phase Diagram for DOTAP:Chol-DNA liposome complexes. Liposomeswere prepared (see Example 1) and complexed with DNA (see Example 2) atvarious concentrations of DNA and liposomes in a final volume of 200 μl.The absorbance at 400 nm was determined for 1:20 dilutions of eachDNA:liposome complex and plotted. An absorbance of 2.0 indicatesprecipitation of the complexes. White boxes indicate data points notdetermined.

FIG. 1B. Production of Chloramphenicol Acetyl Transferase (“CAT”) in themouse lung following systemic delivery using a variety of DNA:liposomeformulations. Each mouse was injected with 82.5 μg plasmid DNA:5 mMliposome (5 mM cationic lipid+5 mM neutral lipid), the optimal DNA:lipidratio for the DDAB (Dimethyldioctadecylammonium Bromide) formulations.CAT expression was normalized to protein concentration in the tissueextracts and is presented as the mean+/−Standard Error of the Mean(“SEM”) of duplicate determinations.

FIG. 2A. CAT production in vivo following systemic delivery ofDOTAP:Chol-DNA complexes. Dose response in the lung using variousDOTAP:Chol-DNA:liposome ratios. The concentrations of DOTAP:Chol variedfrom 0.5 mM to 6 mM complexed with 50 to 175 μg DNA per tail veininjection. (All mM liposome concentrations refer to the cationic lipidor the neutral lipid; therefore, 6 mM DOTAP:Chol=6 mM DOTAP+6 mM Chol.)5 mM DOTAP:Chol was complexed to 50, 100, 125, 150, and 175 μg DNA. At100 μg DNA, DOTAP:Chol was complexed at 5.5 mM, 5 mM, 4.75 mM, 4.5 mM, 4mM, and 3 mM. At 50 μg DNA, DOTAP:Chol was complexed at 6 mM, 5 mM, 4mM, 3 mM, 2 mM, 1 mM, and 0.5 mM. The data points shown here areaverages of CAT production from 40 mouse lungs. Each data point is atleast a duplicate determination, and some data points were averages of 4assays with standard errors varying from 1% to 18% of the mean. Whiteboxes indicate data points not determined.

FIG. 2B. CAT production in various tissues using 3 mM DOTAP:Cholcomplexed with 100 μg DNA per tail vein injection. CAT expression wasnormalized to protein concentration in the tissue extracts and ispresented as the mean+/−SEM.

FIG. 2C. Dose response in the heart using various DNA:liposome ratiosshowing total CAT production. The concentrations of DOTAP:Chol variedfrom 3 mM to 5.5 mM complexed with 100 μg DNA per tail vein injection.

FIG. 2D. Comparison of CAT production levels in the lungs of miceexsanguinated prior to organ harvest and mice that were not bled. Inaddition, whole blood was assayed. CAT activity in the lung is due togene expression in tissue rather than in blood. CAT expression wasnormalized to protein concentration in the tissue extracts and ispresented as the mean+/−SEM.

FIG. 2E. Comparison of CAT production in the lungs of exsanguinated miceusing different DOTAP formulations. All of the following formulationsshown used 4 mM DOTAP: DOTAP:Chol (50:50), DOTAP, DOTAP:Chol:DOPS(50:45:5), DOTAP:DOPE (50:50), and DOTAP:DOPC (50:50). “DOPS” isdioleoyl (18:1) phosphatidyl serine “DOPC” is oleoyl (18:1) phosphatidylcholine (9-cis) octadecanoic acid. All formulations were complexed to100 μg of DNA in a 200 μl final volume. CAT expression was normalized toprotein concentration in the tissue extracts and is presented as themean+/−SEM.

FIG. 3. Cryoelectron micrographs of liposomes and DNA:liposomecomplexes. (A) 5 mM DOTAP:Chol liposomes. A thin film was prepared bydipping and withdrawing a 700 mesh copper grid (3 mm diameter, 3 to 4 μmthick) in the DOTAP:Chol suspension. After blotting away excess liquid,the thin films that form between the bars of the grid were vitrified inmelting ethane. After cryotransfer, the specimen was observed at −170°C. in a Philips CM 12 microscope at low dose, 120 kV (Frederik et al.(1991)). Note the continuity between concentric bilayers in some of thevesicles, giving the shape of a vase with an orifice. (B) 5 mMDOTAP:Chol liposomes mixed with 150 μg DNA/200 μl. These structures arereferred to as DNA-sandwich liposomes. A thin vitrified specimen wasprepared from this DNA:liposome suspension and observed at −170° C. (asin A). Note the packing of DNA (electron opaque addition to the lipid)in the interior of the vesicles. Lipid-DNA interaction has apparentlyresulted in remodeling of the vesicles and changed the accessibility ofDNA. (C) Enlarged image of a DOTAP:Chol vase. (D) Enlarged image of DNApacked between two DOTAP:Chol vases. (E) 5 mM DOTAP:DOPE liposomes. (F)5 mM DOTAP:DOPE liposomes mixed with 150 μg of DNA in a 200 μl finalvolume.

FIG. 4. Proposed model showing cross-sections of DOTAP:Chol liposomesinteracting with supercoiled plasmid DNA. The X indicates fusion oflipid bilayers. The enlarged area shows proposed arrangement of DNAcondensed between two 4 nm bilayers of DOTAP:Chol.

FIG. 5. Production of CAT in the mouse lung following systemic deliveryusing a variety of DOTAP:Chol concentrations complexed to 100 kg of DNAper tail vein injection. Concentrations of DOTAP:Chol used varied from3.0 mM to 5.5 mM. The results showed slightly greater CAT proteinproduction in the lung using 3.0 mM and 4.0 mM liposomes complexed to100 μg DNA per tail vein injection than the amount of CAT produced using5.0 mM liposome complexed to 150 μg DNA. In addition, a dose responsewas produced in vivo following injection of different concentrations ofDOTAP:Chol used to make the DNA:liposome complexes.

FIG. 6. Production of CAT in the mouse lung following systemic deliveryusing 5.0 mM DOTAP:Chol complexed to a variety of DNA amounts per tailvein injection. 5.0 mM DOTAP:Chol may be complexed to a wide range ofplasmid DNA concentration without precipitation of the DNA:liposomecomplexes. Mice were injected with liposomes that contained 50 μg, 100μg, 125 μg, 150 μg and 175 μg of DNA plasmid per tail vein injection. Itis not common for any liposome formulation to be highly tolerant over awide range of DNA:liposome ratios; therefore, our DOTAP:Chol liposomesare extremely unique in this regard. Furthermore, the present inventiondemonstrates a dose response in vivo using different amounts of DNAinjected. At 150 μg DNA, the highest production of CAT was produced inthe lung, and that level of CAT production is up to 150-fold greaterthan any reported in the literature.

FIG. 7. CAT production in the mouse liver at 24 hours after tail veininjection using DNA:liposome complexes coated with succinylatedasialofetuin. Each mouse was injected into the tail vein with 4 mMDOTAP:Chol complexed to 100 μg of DNA in a 200 μl final volume with orwithout addition of succinylated asialofetuin after DNA:liposome mixing.CAT expression was normalized to protein concentration in the tissueextracts and is presented as the mean+/−SEM of duplicate determinations.

FIG. 8. Plot of the radially averaged intensity of DOTAP:Chol-DNAliposome complexes versus scattering wave-vector q. The DOTAP:Chol DNAliposome complexes show the x-ray diffraction maximum, 58.8A, thatconfirms the thickness of DNA+lipid determined by cryo-electronmicroscopy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that liposomes of aspecific composition forming a stable structure are efficient carriersof biologically active agents. The liposome containing one or morebiologically active agents may then be administered into a mammalianhost to effectively deliver its contents to a target cell. The liposomesof the present invention are small and carry a net +/− charge (referredto herein as “ρ”) of about 2 when complexed with a biologically activeagent. The liposomes are capable of carrying biologically active agents,such that the agents are completely sequestered. The liposomes comprisea cationic lipid, DOTAP and cholesterol or a cholesterol derivative.Preferably, at least one biologically active agent to be complexed withthe liposome is negatively charged. Additional biologically activeagents may be complexed with the liposome regardless of their charge, solong as ρ is maintained in the range of 1 to 3, preferably about 2. Theliposome-biologically active agent complex of the present inventionforms an invaginated structure referred to herein as a “sandwichliposome,” because the biologically active agents are sandwiched (andthus sequestered) between the lipid bilayers.

The present invention also provides a targeting means, such that theliposomes can be delivered to specific target sites. The targeting meanscomprises decorating the outside of the sandwich liposome complexes withone or more ligands specific for a particular target site or sites.

“Biologically active agents” as the term is used herein refers tomolecules which affect a biological system. These include molecules suchas proteins, nucleic acids, therapeutic agents, vitamins and theirderivatives, viral fractions, lipopolysaccharides, bacterial fractionsand hormones. Other agents of particular interest are chemotherapeuticagents, which are used in the treatment and management of cancerpatients. Such molecules are generally characterized asantiproliferative agents, cytotoxic agents and immunosuppressive agentsand include molecules such as taxol, doxorubicin, daunorubicin,vinca-alkaloids, actinomycin and etoposide.

The term “nucleic acids” means any double strand or single stranddeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) of variablelength. Nucleic acids include sense and anti-sense strands. Nucleic acidanalogs such as phosphorothioates, phosphoramidates, phosphonatesanalogs are also considered nucleic acids as that term is used herein.Nucleic acids also include chromosomes and chromosomal fragments.Potential genes include but are not limited to: immune system proteinsHLA-B7 and IL-2, cystic fibrosis transmembrane conductance regulator,Factor VIII, Factor IX, insulin and erythropoietin. (Felgner (1997)).

Antisense oligonucleotides may potentially be designed to specificallytarget genes and consequently inhibit their expression. In addition thisdelivery system may be a suitable carrier for other gene-targetingoligonucleotides such as ribozymes, triple helix formingoligonucleotides or oligonucleotides exhibiting non-sequence specificbinding to particular proteins or other intracellular molecules. Forexample, the genes of interest may include retroviral or viral genes,drug resistance genes, oncogenes, genes involved in the inflammatoryresponse, cellular adhesion genes, hormone genes, abnormallyoverexpressed genes involved in gene regulation.

One embodiment of the present invention comprises encapsulating anucleic acid within a liposome and expressing a gene encoded on thenucleic acid within the target host cell, through the use of plasmidDNA. Conversely, the expression of a gene may be inhibited, for example,through the use of antisense oligonucleotides. Alternatively, achemotherapeutic agent may act as the biologically active agent and beencapsulated within a liposome, thereby sequestering its toxic effectsfrom non-targeted tissues.

The present invention may utilize more than one nucleic acid orbiologically active agent in the liposome of the present invention. Forexample, proteins such as DNA binding proteins can be added asadditional biologically active agents to DNA-sandwich liposomes tofacilitate a therapeutic effect. Another example includes sandwichliposomes carrying genes for anti-cancer treatment which are alsocarrying anti-cancer chemotherapeutic agents. This approach isespecially attractive when targeted liposomes are used to deliver bothgene therapy and chemotherapy specifically to cancer cells.

“Liposome” as the term is used herein refers to a closed structurecomprising of an outer lipid bi- or multi-layer membrane surrounding aninternal aqueous space. In particular, the liposomes of the presentinvention form vase-like structures which invaginate their contentsbetween lipid bilayers (see FIG. 4). Liposomes can be used to packageany biologically active agent for delivery to cells. In one example, DNAcan be packaged into liposomes even in the case of plasmids or viralvectors of large size which may be maintained in a soluble form. Suchinvaginated liposome:DNA complexes are ideally suited for directapplication to in vivo systems. These liposomes entrap compounds varyingin polarity and solubility in water and other solvents.

By “nucleic-acid-sandwich” liposomes is meant, the layered compositioncomprising a structure having lipid bilayers with nucleic acid moleculesinserted between and protected by the lipid layers.

One embodiment of the present invention relates to an improved liposomeformulation comprising a nucleic acid, DOTAP and cholesterol whichproduces exceptionally high gene expression and protein production invivo. In addition, these formulations are extremely stable, homogeneousin size, and can complex nucleic acids over a wide range of nucleicacid:liposome ratios. This flexibility allows optimization of thecomplexes for delivery in vivo. Most tissues other than lung areextremely sensitive to the nucleic acid:liposome ratio. In addition, thestability of DOTAP:Chol liposomes at high concentrations of liposome andDNA allows for increased concentrations of DNA for delivery andexpression.

The present invention further relates to a method of preparing thesenovel liposomes comprising the steps of heating, sonicating, andsequential extrusion of the lipids through filters of decreasing poresize, thereby resulting in the formation of small, stable liposomestructures. The method of preparation of the present invention producescomplexes of appropriate and uniform size, which are structurally stableand produce maximal extrusion.

Liposomes comprising DOTAP and at least one cholesterol and/orcholesterol-derivative, present in a molar ratio range of 2.0 mM-10 mMprovide an effective drug delivery system. More preferably, the molarratio of DOTAP to cholesterol is 1:1-3:1. The liposomal composition ofthe present invention has shown to be very stable in a biologicalenvironment.

Cholesterol derivatives may be readily substituted for the cholesterolelement of the present liposome invention. Many cholesterol derivativesare known to the skilled artisan. Examples include but are not limitedto cholesteryl acetate and cholesteryl oleate.

Many DNA preparation protocols are available to the skilled artisan; anyof which can be employed to prepare DNA for use in the liposomes of thepresent invention. Three DNA preparation protocols are preferred,namely, alkaline lysis followed by PEG precipitation, anion-exchangechromatography (Qiagen), and a modified alkaline lysis protocol (seeExample 3). The modified alkaline lysis protocol is a particularlypreferred method to obtain high DNA yield, have low levels of endotoxin,and achieve high levels of gene expression.

Transfer Therapy Methods

The liposomal composition of the present invention may be administeredinto patients parenterally in order to achieve transfer therapy of onenegatively-charged, biologically-active agent along with otherbiologically active agents. Moreover, this technique may be used for “exvivo” transfer therapy where tissue or cells are removed from patients,then treated and finally reimplanted in the patient (U.S. Pat. No.5,399,346, describing the details of ex vivo human gene therapy isincorporated herein by reference). Alternatively, systemic therapy isalso effective in administering the liposome of the present invention.

Many diseases can be treated via the drug delivery system of the presentinvention. Diseases such as diabetes, atherosclerosis,chemotherapy-induced multi-drug resistance, and generally,immunological, neurological (Ho and Sapolsky (1997)) and viral diseases(Friedmann (1997)) can be treated using the present drug deliverysystem.

Non-limiting examples of gene therapy approaches for treating cancerwhich can employ the delivery system of the present invention include:antisense therapy (to block synthesis of proteins encoded by deleteriousgenes), chemoprotection (to add proteins to normal cells to protect themfrom chemotherapies), immunotherapy (to enhance the body's immunedefenses against cancer), pro-drug or suicide gene therapy (to rendercancer cells highly sensitive to selected drugs), tumor suppressor genes(to replace a lost or damaged cancer-blocking gene), antibody genes (tointerfere with the activity of cancer-related proteins in tumor cells)and oncogene down-regulation (to shut off genes that favor uncontrolledgrowth and spread of tumor cells) (Blaese (1997)).

The delivery system of the present invention is also useful forcorrecting the ion transport defect in cystic fibrosis patients byinserting the human CFTR (cystic fibrosis transmembrane conductanceregulator) gene. Oral administration such as nebulization may beparticularly suitable. In addition, the liposomes of the presentinvention can be used for the inhibition of tumor cells by administeringin tumor cells a molecule inhibiting tumorigenesis or a gene coding foran antisense polynucleotide directed to mRNA transcripts of angiogenicfactors. In addition, ribozymes may be encapsulated and enzymaticallyattack specific cellular contents.

The liposomes of the present invention containing the nucleic acid drugcan be administered by intravenous, intramuscular, intraperitoneal,subcutaneous intra-lesional, oral or aerosol means. (Stribling et al.(1992)).

For aerosol administration, a patient receives one or more nasal orbronchial aerosol administrations of the liposome complex. The dosageswill vary based upon age, body composition and severity of disease orcondition. (Caplen et al. (1995)).

Other routes of administration will be known to the skilled artisan andcan be readily used to administer the liposomes of the presentinvention. Examples include but are not limited to mucosal,intra-uteral, intradermal and dermal.

A proposed daily dosage of active compound for the treatment of humansis 0.1 μg DNA/kg to 5.0 mg DNA/kg, which may be convenientlyadministered in 1-10 doses. The actual dosage amount administered can bedetermined by physical and physiological factors such as body weight,severity of condition, idiopathy of the patient and on the route ofadministration. With these considerations in mind, the dosage ofDNA-liposome complex for a particular subject and/or course of treatmentcan readily be determined.

The liposome of the invention may be formulated for parenteraladministration by bolus injection or continuous infusion. Formulationfor injection may be presented in unit dosage form in ampoules, or inmulti-dose containers with an added preservative. The compositions maytake such forms as suspension, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for reconstitution with a suitablevehicle, e.g. sterile pyrogen-free water, before use.

The liposomes according to the invention may be formulated foradministration in any convenient way. The invention therefore includeswithin its scope pharmaceutical compositions comprising at least oneliposomal compound formulated for use in human or veterinary medicine.Such compositions may be presented for use with physiologicallyacceptable carriers or excipients, optionally with supplementarymedicinal agents. Conventional carriers can also be used with thepresent invention.

For oral administration, the pharmaceutical composition may take theform of, for example, tablets, capsules, powders, solutions, syrups orsuspensions prepared by conventional means with acceptable excipients.

An improved method for preparing the liposomes of the present inventionemploys sonication, heating, and extrusion (see Example 1 for detaileddescription). Generally, the method requires that the lipid componentsbe mixed in the appropriate concentrations, dissolved in an organicsolvent, such as chloroform or the like, evaporated into a thin film andlyophilized. The film is then rehydrated in an aqueous solution andmixed for a period at 35°-60° C. Thereafter, the mixture is sonicatedand heated to a temperature between 40° C.-60° C. This mixture is thensequentially extruded through filters of decreasing size. Sonicatedliposomes are preferably extruded through filters of decreasing poresizes, including 0.1 μm, by using sufficient heating.

Various nucleic acids may be added to these liposomes in a wide range ofconcentrations. Nucleic acids are preferably added to the liposomes at aconcentration of 50-300 μg per dose. These concentrations vary widelydepending upon the ratio of DOTAP:Chol in the particular liposomepreparation. For example, if a liposome having a molar ratio ofDOTAP:Chol of about 1:1 is used, then a preferred concentration ofnucleic acid is between 80-175 μg per dose.

Extruded DOTAP:Chol liposomes prepared by the method of the presentinvention were compared to non-extruded DOTAP:Chol liposomes(multilamellar vesicles, MLVs) prepared by the conventional method. (Liuet al. (1997)). The extruded liposomes form sandwich liposomes whereasthe non-extruded liposomes do not form such structures. Proteinexpression from nucleic acid: sandwich liposomes was approximately2-fold higher as compared to MLVs. Furthermore, MLVs containing 5 mM to10 mM DOTAP:Chol have increased toxicity when delivered systemically.

Production of chloramphenicol acetyl transferase (“CAT”) is the mostwidely used method to measure protein expression and is a wellrecognized model protein expression system. (Wheeler et al. (1996); Leeet al. (1996); Felgner (1996)). In one embodiment of the presentinvention the sandwich liposome complexes (i.e. FIGS. 2A, D, E) produced100-fold greater amounts of CAT in the lung compared to CAT producedusing DOTIM:Chol SUVs. (Solodin et al. (1995)). Those skilled in the artwill readily recognize that any nucleic acid can be used in the presentinvention.

By using sandwich liposomes for systemic nucleic acid delivery, a broadbiodistribution is produced (i.e. FIG. 2B) that is greater than thatproduced by any other cationic liposome formulation reported. (Zhu etal. (1993); Philip et al. (1993); Solodin et al. (1995); Liu et al.(1995); Thierry et al. (1995); Tsukamoto et al. (1995); Aksentijevich etal. (1996); Wheeler et al. (1996); Liu et al. (1997); Hong et al.(1997); Lee et al. (1996); Felgner (1996)).

Targeted Delivery

Because the biologically active agent is sequestered in the sandwichliposomes, targeted delivery is achieved by the addition of peptides andother ligands without compromising the ability of these liposomes tobind and deliver large amounts of the agent. The ligands are added tothe liposomes in a simple and novel method. First, the lipids are mixedwith the biologically active agent of interest. Then ligands are addeddirectly to the sandwich-liposomes to decorate their exterior surface.The stability and net positive charge of the liposomes allow ligands tobe directly added to their exterior.

The sandwich liposome complexes of the present invention may be used tomake effective artificial viruses. Because the outside of the sandwichliposome complexes is substantially free of biologically active agents,targeting ligands may be placed on the outside after sandwich liposomeformation, without compromising the effect of the targeting ligand orthe ability of the biologically active agents to be delivered andexpressed. This may enable delivery to specific organs and tissues. Thesize of the sandwich liposome complexes responsible for efficientdelivery, 200 to 450 nm (see also Table 1), is preferred for theaddition of targeting ligands. Our experiments demonstrate theusefulness of this approach (See Example 9, FIG. 7).

Many ligands may be employed for this targeting step of liposomepreparation, depending upon the site targeted for liposome delivery. Forexample, lactosyl ceramide, and peptides that target the LDL receptorrelated proteins, such as apolipoprotein E3 (“Apo E”) has been useful intargeting the liposomes of the present invention to the liver. Inparticular, the use of the Apo E ligand resulted in a 4-fold greatergene expression in the liver.

In addition, magnetic resonance imaging shows that the half-life ofsandwich liposome complexes is at least 5 hours in circulation. Thisdemonstrates that the sandwich liposomes have time to reach the intendedtarget intact.

Alternatively, monoclonal antibody fragments may be used to targetdelivery to specific organs in the animal including brain, heart, lungsor liver.

The present method to add ligands only to the outer surface aftercomplex formation is unique and has the advantage of avoiding disruptionof the biologically active agent liposome complex formation due tosteric or ionic interactions with the targeting ligand.

One embodiment of the liposomes of the present invention are completelyinvaginated with two concentric lamellae and a small orifice with anapproximate diameter of 50 nm (FIG. 3A), which are called a “vasestructure” (FIG. 3C). For example, of 536 vesicles observed, 88% wereinvaginated structures, and only 12% were the typical small unilamellarvesicles (SUVs). The invaginated sandwich liposomes of the presentinvention include other structures, such as, bilamellar vases,bilamellar and unilamellar tubular shapes, unilamellar erythrocyteshapes, and unilamellar bean shapes.

Liposomes are examined after each step in the process of making thesandwich liposomes and unilamellar spheres are observed until extrusionthrough the 0.1 μm filter. This extrusion step produces invaginatedstructures with excess surface area. Performing only mild sonication(see Example 1) prior to extrusion is also recommended because highfrequency sonication of DOTAP:Chol liposomes produces only SUVs andmicelles. The bulk (88%) of sandwich liposomes prepared by the instantmethod are bilamellar and unilamellar invaginated vesicles.

It appears that the biologically active agent adsorbs onto theinvaginated and tubular liposomes via electrostatic interactions (FIG.4). Attraction of a second liposome to this complex results in furthercharge neutralization. If the liposomes are of unequal size, expandingelectrostatic interactions with the biologically active agent causeinversion of the larger liposome and total engulfment of thebiologically active agent. These structures are sandwich liposomes.Inversion can occur in these liposomes because of their excess surfacearea, which allows them to accommodate the stress created by the lipidinteractions with the biologically active agent.

For example, DNA binding reduces the surface area of the outer leafletof the bilayer and induces the negative curvature due to lipid orderingand reduction of charge repulsion between cationic lipid headgroups.Condensation of the internalized lipid sandwich expands the spacebetween the bilayers and may induce membrane fusion to generate theapparently closed structures in FIGS. 3B, D. Interaction of more thantwo liposomes may create the more complex structures seen in thismicrograph. The predicted thickness of 10.5 nm for DNA sandwichedbetween 2 bilayers (FIGS. 3D, G) is in agreement with observedmeasurements of these areas as shown in FIG. 3B. In addition, thethickness of DNA+lipid is confirmed by small angle x-ray scatteringanalyses (FIG. 8).

Alloying soft bilayers that contain dioleoyl chains with cholesterol isknown to increase the stretching elastic modulus by up to an order ofmagnitude (Lasic and Needham (1995)). Liposomes that have mechanicallyweaker bilayers cannot efficiently undergo an inversion, and the agentcomplexed to these liposomes is less protected in the circulation. Thepresence of appropriate levels of cholesterol in the bilayer providessufficient strength to liposomes for efficient “vase” formation. Thesize, mechanical strength, and flexibility of the lipid vesicles as wellas the biologically active agent-liposome ratio are critical for thisself-assembly mechanism of DNA condensation on the interior ofinvaginated liposomes. This model predicts that an approximate ρ valueof 2 will be preferred to neutralize all charge associated with thebiologically active agent by generating the lipid bilayer “sandwich”.This also demonstrates that the outside of the sandwich liposomecomplexes will be positively charged and free of biologically activeagent.

The present invention clearly shows that cholesterol is an efficientneutral lipid in a liposome complex for in vivo DNA delivery. Highcontent of cholesterol is known to increase the stability of liposomes.The presence of cholesterol stabilizes bilayers and complexes in theplasma against mechanical breakage upon adsorption of plasma components.In addition, DOTAP is an effective cationic lipid. The combination ofDOTAP:Chol to produce cationic liposomes under the specific method ofthe present invention resulted in liposomes with unique and usefulproperties for in vivo gene delivery. The present invention provides anup to 150-fold improvement in gene expression in several organs usingextruded DOTAP:Chol liposomes for systemic DNA delivery as compared withprior art techniques. This vast improvement allows increased efficiencyof gene transfer in vivo.

Unlike other cationic liposomes, DOTAP:Chol liposomes, in oneembodiment, form stable DNA complexes over a broad range of DNA:liposomeratios. This flexibility allows optimization of the complexes for invivo delivery to different tissues. Most tissues other than lung arevery sensitive to this ratio (corresponding to ρ=2); therefore, thisratio must be carefully optimized for each DNA concentration. Thestability of DOTAP:Chol at high concentrations of liposome and DNAallows for increased concentrations of DNA to be delivered andexpressed. An effective liposome system must protect DNA in thecirculation, yet be able to deliver the DNA effectively to tissues.These properties have been achieved with the DOTAP:Chol liposomes as aresult of their more cohesive bilayer and their ability to internalizeand therefore protect DNA.

The following examples serve to illustrate further the present inventionand are not to be construed as limiting its scope in any way.

While the invention is described above in relation to certain specificembodiments, it will be understood that many variations are possible,and that alternative materials and reagents can be used withoutdeparting from the invention. In some cases such variations andsubstitutions may require some experimentation, but will only involveroutine testing.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention and others can, by applyingcurrent knowledge, readily modify and/or adopt for various applicationssuch specific embodiments without departing from the generic concept,and therefore such adaptations and modifications are intended to becomprehended within the meaning and range of equivalents of thedisclosed embodiments.

All of the references mentioned in the present application areincorporated in toto into this application by reference thereto.

EXAMPLE 1

DOTAP:Chol liposomes, as well as other liposome formulations, wereprepared using the following procedure: the cationic lipid (DOTAP orDDAB) was mixed with the neutral lipid (Chol or DOPE) at equimolarconcentrations. The mixed powdered lipids were dissolved in HPLC-gradechloroform (Mallinckrodt) in a 1 L round bottomed flask. The clearsolution was placed on a Buchi rotary evaporator at 30° C. for 30 min tomake a thin film. The flask containing the thin lipid film was driedunder vacuum for 15 min. The film was hydrated in 5% dextrose in water(“D5W”) to give a final concentration of 20 mM DOTAP (or 20 mM DDAB) and20 mM Chol (or 20 mM DOPE), and is referred to as 20 mM DOTAP:Chol. Thehydrated lipid film was rotated in a 50° C. H₂O bath for 45 min and thenat 35° C. for an additional 10 min. The mixture was allowed to stand inthe parafilm-covered flask at room temperature overnight. On thefollowing day, the mixture in the flask was sonicated for 5 min at 50°C., transferred to a tube, and was heated for 10 min at 50° C. Themixture was sequentially extruded through decreasing size filters: 1 μm,0.45 μm, 0.2 μm, and 0.1 μm (Whatman) using syringes. Whatman Anotopfilters, 0.2 μm and 0.1 μm, were used. Portions of the liposome mixturethat did not pass through the first 0.1 μm filter, were heated again at50° C. for 5 min before passing through a new 0.1 μm filter. Filteredfractions were pooled and stored under argon gas at 4° C. DOTAP and DOPEwere purchased from Avanti Polar Lipids. DDAB was purchased from SigmaChemical Company, and highly purified cholesterol was purchased fromCalbiochem.

EXAMPLE 2 DNA-Sandwich Liposomes

DNA:liposome complexes were prepared the day prior to their use in ananimal host. DNA was diluted in D5W (5% dextrose in water), and stockliposomes were diluted in D5W to produce various ratios ofDNA:liposomes. The final volumes of both the DNA solution and theliposome solution used for mixing were equal. Dilutions and mixings wereperformed in 1.5 ml Eppendorf tubes with all reagents at roomtemperature. The DNA solution was added rapidly at the surface of theliposome solution using a pipette tip. The DNA:liposome mixture wasmixed rapidly up and down twice using a Pipetman. DNA:liposome complexeswere stored overnight at 4° C.

The protocol used for DNA preparation, referred to herein as the Debsprotocol, is a variation of the alkaline lysis procedure described byManiatis (Sambrook et al. (1989)), and includes a 2 hour Proteinase Kdigestion step immediately following RNase A digestion. This methodconsistently produced about 20-fold greater amounts of DNA than thealkaline lysis procedure followed by polyethylene glycol (PEG)precipitation, and yields were in the range of 10 to 28 mg of plasmidDNA per liter of bacterial culture. This DNA preparation protocolresulted in no toxicity of DNA:liposome complexes in mice. Endotoxinlevels of DNA prepared by the different methods were determined usingthe chromogenic limulus amebocyte lysate assay (Kinetic-QCL;BioWhittaker). Endotoxin levels of 120 Endotoxin Units (EU)/μg DNA, 16EU/μg DNA, and 8 EU/μg DNA were determined for the Maniatis alkalinelysis method followed by PEG precipitation, the Qiagen Maxi-prep Kit,and the Debs protocol for preparation of plasmid DNAs, respectively. Nogenomic DNA, small DNA fragments, or RNA were detected in the DNAprepared by the Debs protocol, and the OD_(260/280) ratios of allplasmid DNA preparation were 2.0.

EXAMPLE 3 In Vivo Administration of DNA-Sandwich Liposomes

For in vivo intravenous administration, 6-weeks old (˜20 g) BALB/c micewere injected in the tail vein with 200 μl of DNA:liposome complexesusing a 27-gauge syringe needle. Samples were placed at room temperaturefor 1 hour prior to tail vein injection. Mice were sacrificed 24 hourspost-injection, and the organs were harvested and quickly frozen onliquid nitrogen. Tissue extracts were prepared as previously described(Stribling et al. (1992)). Lymph node extracts consisted of pooledmesenteric, axillary, iliac, submandibular, and inguinal lymph nodes.ELISAs were performed using the Boehringer Mannheim CAT ELISA kit. AllCAT protein determinations were corrected for any CAT immunoreactivitydetected in control tissues, and the lowest levels of CAT proteinreported for any experimental tissues were at least 3-fold higher thanbackground. Protein determinations were performed using the Micro BCAkit (Pierce). This work was conducted in accordance with NCI/FCRDCguidelines using an approved animal protocol.

In order to demonstrate that the DNA is protected within the liposome,DNA-sandwich liposomes can be subjected to filtration throughpolysulfone filters of various pore sizes. The liposomes maintain fullactivity through the 1.0 μm and 0.45 μm filters, demonstrating that theDNA is fully sequestered and protected from the exterior. (See Table 1)If the DNA had been attached to the outside of the liposome, its proteinexpression activity would have been lost by filtering, since polysulfonestrips DNA from complexes carrying it on the outside.

Table 1 also illustrates that the DNA-sandwich liposomes capable ofefficient DNA delivery are preferably larger than 200 nm in size.Particularly preferred DNA-sandwich liposomes include those between 200nm and 450 nm in size. The exact size may vary with the DNA containedtherein, the route of administration and the condition being treated.The skilled artisan can readily determine the appropriate size complexesbased upon these parameters.

TABLE 1 CAT production in organs using size fractionated DNA: liposomecomplexes Total CAT protein produced (ng)* No 1.0 μm 0.45 μm 0.2 μmOrgan Filtration Filtration Filtration Filtration Lung 168.6 207.0 154.868.4 Heart 8.5 7.6 6.6 1.8 Liver 5.5 4.6 6.0 2.4 Muscle (quadricep) 5.06.0 5.7 1.5 Kidney 1.0 1.6 1.2 0.6 Thymus 0.9 0.8 1.2 0.1 Colon 0.4 0.70.9 0.6 Spleen 0.2 0.2 0.3 0.1 Lymph nodes 0.1 0.1 0.1 0.03 Brain 0.10.1 0.2 0.1 *Total CAT protein was determined by ELISA for organsharvested 24 hours post-injection. Mice were injected with 3 mMDOTAP:Chol + 100 ug of CAT plasmid DNA filtered through the indicatedpore size after liposome:DNA mixing. Results are the mean of duplicateanalyses determined using two animals in each group.

EXAMPLE 4 Comparison of Liposome Combinations

In order to demonstrate the unexpected nature of the present invention,the capacity of different liposome formulations to form complexes withDNA was examined. Various concentrations of liposomes were mixed withDNA in a final volume of 200 μl, and the absorbance at 400 nm wasdetermined for 1:20 dilutions of each sample. The DNA:liposome phasediagram showed that DOTAP containing liposomes stably complexed largeamounts of DNA over a wide range of DNA:liposome ratios (FIG. 1A).Soluble complexes using DDAB liposomes had lower maximal DNAconcentrations and were stable over a narrow range using either DOPE orChol. A maximum of 82.5 μg of DNA was optimally complexed with 5 mM DDABformulations in a 200 μl final volume without causing precipitation.

On the basis of their physicochemical properties, these liposomeformulations were complexed to DNA and introduced into mice by tail veininjection. Systemic DNA delivery and gene expression were compared using1:1 formulations of DDAB:Chol, DDAB:DOPE, DOTAP:Chol, and DOTAP:DOPEliposomes, each at 5 mM, complexed to 82.5 μg plasmid DNA in a 200 μlfinal volume. This DNA:liposome ratio was found to be optimal for invivo expression of DNA delivered by DDAB:Chol liposomes (Liu et al.(1995)). At 24 hours postinjection the mice were sacrificed, and thelevels of CAT production in the lung were determined (FIG. 1B). Bothextruded and non-extruded preparations of the above lipid formulationswere injected into mice and the results compared to each other and tothose obtained by other investigators. The non-extruded liposomepreparations resulted in lower expression in all cases compared to thecorresponding extruded preparations. CAT production using DDAB:Cholliposomes prepared by sonication without extrusion, was in excellentagreement with CAT production reported using the same conditions (Liu etal. (1995)), based on the specific activity of CAT at 100,000 U per mgper min.) and the identical plasmid (The CAT plasmid used in allexperiments were p4119 (Liu et al. (1995)). Using additional heating andextrusion steps (see Example 1) in the preparation of DDAB:Cholliposomes prior to mixing with DNA, increased expression 2-fold.Interestingly, the extruded DOTAP:Chol-DNA liposome complexes produced50-fold greater amounts of protein expression in the lung compared tothe highest levels that have been reported using sonicated DDAB:Chol inthe same tissue (Liu et al. (1995)). In addition, the level of CATprotein produced in the lung using this novel formulation was greaterthan 50-fold compared to that using sonicated DOTMA:DOPE (DOTMA isN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (alsoknown as Lipofectin™)) (Zhu et al. (1993)) or sonicated DOTIM:Chol(DOTIM is1-[2-(9(Z)-Octadecenoyloxy)ethyl]-2-(8(Z)-heptadecnyl)-3-(2-hydroxyethyl)imidazoliniumchloride) (Solodin et al. (1995)). Extruded DOTAP:DOPE-DNA liposomecomplexes and extruded DDAB:DOPE-DNA liposome complexes produced farless CAT in the lungs compared to DOTAP:Chol-DNA liposome complexes.

To determine whether other extruded DOTAP:DOPE formulations wouldimprove delivery by using the optimal DNA:liposome ratio for the lungs,4 mM DOTAP:DOPE complexed to 100 μg of DNA were injected into mice. CATproduction remained 50-fold lower than that observed with 4 mMDOTAP:Chol-DNA liposome complexes (FIG. 2E). In addition, CAT productionwas determined using other extruded liposome formulations including 4 mMDOTAP-DNA liposome complexes, 4 mM DOTAP:Chol: DioleoylPhosphatidylserine (DOPS)-DNA liposome complexes, and 4 mM DOTAP:OleoylPhosphatidylcholine (DOPC)-DNA liposome complexes. As shown in FIG. 2E,DOTAP and all other formulations were not as effective as DOTAP:Chol forgene expression in vivo, and these formulations did not provide as broada biodistribution as that using DOTAP:Chol (data not shown). Addition ofsmall amounts of DOPS, 5%, to DOTAP:Chol (50:45) dramatically reduced invivo gene expression (FIG. 2E), and use of DOPC instead of Choleliminated in vivo gene expression almost entirely (FIG. 2E). Both DOPSand DOPC contain large headgroups and could perhaps interfere with theDNA:liposome assembly shown in our model (FIG. 4).

EXAMPLE 5

In order to evaluate in vivo expression, 100 μg of DNA were mixed withvarying concentrations of DOTAP:Chol. The highest levels of CAT wereproduced in lungs using 3 mM and 4 mM DOTAP:Chol complexed to 100 μgDNA, with an approximate +/− charge ratio ρ=2. However, significantexpression was obtained at all ratios (FIG. 2A). Furthermore, themaximal expression using 100 μg DNA was greater in lungs than thatproduced from injections of 5 mM DOTAP:Chol complexed to 150 μg DNA, andthis expression was approximately 100-fold greater than previouslyreported using other cationic liposomes (Zhu et al. (1993); Philip etal. (1993); Solodin et al. (1995); Liu et al. (1995) based on thespecific activity of CAT at 100,000 U per mg per min.). In addition,complexes of 3 mM to 4 mM DOTAP:Chol mixed with 100 μg DNA were stableupon storage at 4° C. for longer periods than complexes mixed with 125μg and 150 μg DNA at optimal DNA:liposome ratios. To determine whetherDOTAP:DOPE formulations would improve delivery by using the optimalDNA:liposome ratio, 3 mM DOTAP:DOPE complexed to 100 μg DNA wereinjected. However, the levels of CAT produced remained 50-fold lowerthan that produced by 3 mM DOTAP:Chol-DNA liposome complexes.

EXAMPLE 6

Using different concentrations of DOTAP:Chol complexed to 50 μg of DNA,there were more dramatic differences in the dose-response in the lung ascompared to formulations containing higher amounts of DNA. Optimalproduction of CAT in the lung was observed after injection of complexescontaining 2 mM DOTAP:Chol complexed to 50 μg of DNA (FIG. 2A). Thus,optimal expression at all DNA concentrations was achieved at comparableDNA:liposome ratios namely approximate +/− charge ratio ρ=2. However,the lower colloidal concentration of DNA and liposomes using 50 μg DNAreduced the expression levels even at the optimal DNA:liposome ratio. Inaddition, CAT production fell sharply at DNA:liposome ratios eitherlower or higher than the optimum.

To assess the amount of protein expressed in each tissue, total proteinproduction was calculated for animals receiving 3 mM or 4 mM DOTAP:Cholcomplexed to 100 μg DNA (Table 2). Data showed that high levels of geneexpression could be obtained using DOTAP:Chol as a systemic deliveryvehicle. Although expression in lung was relatively insensitive to theratio of DNA:liposome, most other tissues showed a 2 to 4-fold decreasein protein production using 4 mM instead of 3 mM DOTAP:Chol. Thus, thegreater sensitivity to DNA:liposome ratio noted for heart in FIG. 2C mayapply to most tissues other than the lung.

TABLE 2 Effect of DNA: lipid ratio on organ distribution of proteinproduction. Total CAT Protein produced (ng)* 4 mM 3 mM DOTAP: DOTAP:Organ Chol Chol Lung 170.4 166.8 Heart 8.7 3.5 Liver 5.6 3.4 Muscle(quadricep) 5.1 1.8 Kidney 0.9 0.6 Colon 0.4 0.1 Spleen 0.3 0.1 LymphNodes 0.2 0.1 Thymus 0.2 0.1 Brain 0.2 0.1 *Total CAT protein wasdetermined by ELISA for organs harvested 24 hours post-injection. Micewere injected with 100 μg CAT plasmid complexed with the indicatedconcentrations of DOTAP: cholesterol. Results are the mean of duplicateanalysis determined using two animals in each group.

EXAMPLE 7

Because the in vitro data showed that DOTAP:Chol formed stable colloidalcomplexes over a wide range of DNA:liposome ratios (FIG. 1A), differentgroups of mice were injected with complexes consisting of 0.5 mM to 6 mMDOTAP:Chol mixed with 50 to 175 μg DNA in a 200 μl final volume. Thegoal was to determine the optimal DNA:liposome ratio for the DOTAP:Cholsystem. Results from initial experiments showed that DNA delivery andgene expression were further increased by optimizing this DNA:liposomeratio. Maximal CAT production in mouse lung was produced by the use of3-4 mM DOTAP:Chol complexed to 100 μg DNA (corresponding to a +/− chargeratio {ρ} of 2, FIG. 2A). The highest level of CAT production wasapproximately 100-fold greater than previously achieved (Zhu et al.(1993); Philip et al. (1993); Solodin et al. (1995); Liu et al. (1995)).The CAT activity was based on the specific activity of CAT at 100,000 Uper mg per min. Although higher amounts of DNA formed stable complexeswith these liposomes, toxicity was induced after 200 μl injections of250 and 300 μg DNA complexed with 9 mM and 10 mM DOTAP:Chol,respectively.

EXAMPLE 8 Tissue Expression Levels

Using the optimal DNA:liposome ratio and colloidal concentration for theDOTAP:Chol system identified in these experiments, (3 mM DOTAP:Cholcomplexed to 100 μg DNA), CAT production in other tissues was studied(FIG. 2B). Significant amounts of CAT were produced in all tissuesexamined (FIG. 2B). Approximately 75 to 150-fold greater amounts of CATwere produced in the lung, heart, liver, muscle, and kidney thanpreviously reported using other cationic lipids (Zhu et al. (1993);Philip et al. (1993); Solodin et al. (1995); Liu et al. (1995)) based onthe specific activity of CAT at 100,000 U per mg per min. Expression inthe heart was optimal at 3 mM DOTAP:Chol+100 μg DNA and decreasedmarkedly at other ratios (FIG. 2C). For lymph nodes and spleen, thelevels of CAT protein produced were about 25-fold and 2-fold greaterthan prior reports, respectively. CAT production in all tissues wasdetermined for every formulation. It was found that CAT production wasoptimal using 3 mM DOTAP:Chol complexed to 100 μg DNA in all tissuesexcept for lymph nodes. Gene expression in lymph nodes was optimal using5 mM DOTAP:Chol complexed to 150 μg DNA, and CAT production wasincreased 75-fold over that previously reported. Quantitation of CATproduction has not been previously reported in the thymus, skin, tail,colon, and brain, although expression has been noted in some of thesetissues (Zhu et al. (1993); Philip et al. (1993); Solodin et al. (1995);Liu et al. (1995); Thierry et al. (1995)).

If nucleated cells in the blood were transfected by the systemicallydelivered complexes, expression of CAT in some tissues may result fromblood included in the specimens. This was not the case in the lung,because CAT production in whole blood, 47 pg/mg total protein, was muchlower than in the lungs obtained from the same mice (FIG. 2D).Furthermore, CAT concentration measured in the lungs of exsanguinatedmice was elevated about 30%, because the total protein levels were lowerin the absence of blood. CAT levels for most other tissues from theexsanguinated mice showed either increased concentrations of CAT orconcentrations similar to nonbled mouse tissues. CAT concentrationlevels decreased in the tail, skin, and brain for samples assayed fromexsanguinated mice, suggesting that CAT production detected in thesetissues may, in part, be contributed by nucleated blood cells.

EXAMPLE 9 Targeting Ligands

Succinylated asialofetuin was made as previously described (Kaneo et al.(1991)), and 4 mM DOTAP:Chol was complexed with 100 μg of DNA in a 200μl final volume. These complexes were filtered through a 0.45 μmpolysulfone filter (Whatman). Succinylated asialofetuin, to yield afinal concentration of 0.2 mg/ml, was added to these filteredDNA:liposome complexes using a Pipetman pipet tip. This mixture wasmixed slowly up and down twice in the pipet tip. DNA:liposome complexeswere stored overnight at 4° C. No precipitation of theDNA:liposome-asialofetuin complexes occurred. These complexes wereinjected into the tail vein of mice as described above.

Succinylated asialofetuin is highly negatively-charged; therefore, itcan bind tightly to the positively charged surface of liposomes thatcontain DNA between the invaginated liposomes. There was adose-dependent rise in the OD₄₀₀ after addition of succinylatedasialofetuin. The absorbance at 400 nm was determined for 1:20 dilutionsof the following DNA:liposome complexes with or without succinylatedasialofetuin. DOTAP:Chol-DNA liposome complexes had an OD₄₀₀ of 0.815;and DOTAP:Chol-DNA+0.1 g/ml succinylated asialofetuin andDOTAP:Chol-DNA+0.2 mg/ml succinylated asialofetuin had OD₄₀₀ readings of0.844 and 0.873, respectively. Addition of succinylated asialofetuin atamounts greater than 0.2 mg/ml produced immediate precipitation ofDNA:liposome complexes. These observations show that succinylatedasialofetuin strongly interacts with the DOTAP:Chol-DNA liposomecomplexes. Succinylated asialofetuin was added to the surface of theliposome complexes to achieve greater gene expression in the liver.Asialofetuin is an asialoglycoprotein containing terminal galactosylresidues and has been used to efficiently target liposomes to the liver.(Spanjer and Scherphof (1983); Spanjer et al. (1984); Dragsten et al.(1987); Murahashi and Sasaki (1996); Hara et al. (1995)). In addition,changing the surface charge on the outside of the DNA:liposome complexesto reduce ionic interactions with endothelial proteoglycans (Mislick andBaldeschwieler (1996)) may also facilitate organ-specific delivery.

Addition of succinylated asialofetuin to preformed DNA:liposomecomplexes provided a ligand for the hepatic asialoglycoprotein receptor(Ashwell and Harford (1982)) and increased CAT production in the liverseven-fold (FIG. 7). This targeting was specific for the liver, as CATexpression in other organs shown in FIG. 2B was not increased. Thiswidely used ligand was employed solely to demonstrate feasibility of thepresent method for adding ligands.

EXAMPLE 10 Cyro-Electron Microscopy

Thin films were prepared by dipping and withdrawing a 700-mesh coppergrid (3 mm diameter, 3 to 4 μm thick) in the liposome or theDNA:liposome suspensions. After excess liquid was removed by blotting,the thin films that formed between the bars of the grid were vitrifiedin melting ethane. After cryotransfer, the specimen was observed at−170° C. in a Philips CM 12 microscope at low dose, 120 kV 19.

To examine the mechanism of the high in vivo gene delivery, theDNA-sandwich:liposome complexes were examined by studying theirmorphology as well as that of non-DNA associated liposomes withcryo-electron microscopy. Extruded DOTAP:Chol liposomes in the absenceof DNA showed many completely invaginated liposomes with two concentriclamellae and a small orifice. Most liposomes were spherical with anapproximate diameter of 50 nm.

Vase structures represented one-third of the entire liposome population.Large tubular structures were also observed, explaining the somewhatlarger size of approximately 240 nm, determined by dynamic lightscattering using a Coulter N4 particle size analyzer.

When these liposomes were complexed with DNA at optimal concentrations,with a +/− charge ratio ρ=2 (Lasic et al. (1997)), the DNA was localizedto the interior of the liposomes (FIG. 3B). DOTAP:DOPE (dioleoylphosphatidyl-ethanolamine) and DOTAP:Chol complexes with DNA were turbidcolloidal solutions with mean particle size of 445 nm and 405 nm,respectively. Particle size did not depend on dilution, and turbidityobeyed Beer Lambert Law indicating stability of these complexes invitro. DOTAP:DOPE liposomes also form “vase structures”; however, theorifices were larger and many spheres were formed (FIG. 3E). Inaddition, there were many structures with little or no DNA assembled inthe extruded DOTAP:DOPE liposomes (FIG. 3F), and the DNA was frequentlyfound on the outside of these liposomes (FIG. 3F). DDAB:DOPE andDDAB:Chol liposomes did not form “vase structures”. The internalizationof DNA within “vases” is a unique feature of extruded DOTAP liposomesand have not been observed for any other DNA:liposome complex studied bycryo-electron microscopy (Frederik et al. (1991)). The “vase structures”observed for DOTAP:Chol may contribute to the high systemic delivery andgene expression achieved with these formulations.

Small Angle X-ray Scattering (SAXS)

DOTAP: Chol-DNA complexes were concentrated into a highly orderedstructure by centrifugation or drying, and both techniques produced thesame SAXS results. SAXS experiments were performed using a rotatinganode x-ray source GX-13 (Elliot, England) focused by two bent x-raymirrors. The 2-dimensional x-ray pictures were taken with a Franck-typecamera at 15 cm distance from the sample, using Kodak phosphorousscreens (Kodak, N.Y.) that were scanned by an image plate reader(PhosphorImager SI, Molecular Dynamics, CA). The radial intensityaverages were determined using our modification of the NIH-Image 1.57image processing program (Wayne Rasband, National Institutes of Health,MD). The results are shown in FIG. 8.

REFERENCES

Aksentijevich, I. et al. Human Gene Ther. 7, 1111 (1996).

Ashwell, G. and Harford, J. Annu. Rev. Biochem. 51, 531 (1982).

Blaese, R. M. Scientific American 6, 111 (1997).

Caplen, N. J. et al. Nature Medicine 1(1), 39-46 (1995).

Dragsten, P. R. et al. Biochim. Biopys. Acta 926, 270 (1987).

Felgner, J. H. et al. J. Biol. Chem. 269, 2550 (1994).

Felgner, P. L. et al. Proc. Natl. Acad. Sci. USA 84, 7413 (1987).

Felgner, P. L. Hum. Gene Ther. 7, 1791 (1996).

Felgner, P. L. Scientific American 6, 102 (1997).

Frederik, P. M. et al. J. Microscopy 161, 253 (1991).

Friedmann, T. Scientific American 6, 96 (1997).

Gao X. et al. Biochem. Biophys. Res. Commun. 179, 280 (1991).

Gustafsson, J. et al. Biochim. Biophys. Acta 1235, 305 (1995).

Hara, T. et al. Gene Therapy 2, 784 (1995).

Ho, D. Y. and Sapolsky, R. M. Scientific American 6, 116 (1997).

Hong, K. et al. FEBS Lett. 400, 233 (1997).

Kaneo, Y. et al. Chem. Pharm. Bull. 39, 999 (1991).

Lasic D. D. and Needham, D. Chemical Reviews 95, 2601 (1995).

Lasic D. D. et al. J. Am. Chem. Soc. 119, 832 (1997).

Lee, E. R. et al. Hum. Gene Ther. 7, 1701 (1996).

Liu, Y. et al. J. Biol. Chem. 270, 24864 (1995).

Liu, Y. et al. Nature Biotech. 15, 167 (1997).

Mislick, K. A. and Baldeschwieler, J. D. Proc. Natl. Acad. Sci. USA 93,12349 (1996).

Murahashi, N. and Sasaki, A. Biol. Pharm. Bull. 19, 418 (1996).

Nabel, E. G., et al. Science 249, 1285 (1990).

Philip, R. et al. J. Biol. Chem., 268, 16087 (1993).

Radler, J. O. et al. Science 275, 810 (1997).

Sambrook J. et al. Molecular Cloning, 2nd Edition, pp. 1.38-1.39 (1989).

Solodin, I. et al. Biochemistry 34, 13537 (1995).

Soriano P., et al. Proc. Natl. Acad. Sci. USA 80, 7128-7131 (1983).

Spanjer, H. H. and Scherphof, G. L. Biochim. Biopys. Acta 734, 40(1983).

Spanjer, H. H. et al. Biochim. Biopys. Acta 774, 49 (1984).

Stamatatos, L. et al. Biochemistry 27, 3917 (1988).

Stribling R. et al. Proc. Natl. Acad. Sci. USA 89, 11277 (1992).

Thierry, A. R. et al. Proc. Natl. Acad. Sci. USA 92, 9742 (1995).

Tsukamoto, M. et al. Nature Genetics 9, 243 (1995).

Wang, C. et al. Proc. Natl. Acad. Sci. USA 84, 7851 (1987).

Wheeler, C. J. et al. Proc. Natl. Acad. Sci. USA 93, 11454 (1996).

Xu, Y. and Szoka, F. C. Biochemistry 35, 5616 (1996).

Zhu, N. et al. Science 261, 209 (1993).

We claim:
 1. An invaginated vase-like liposome produced by the stepscomprising: i) heating 1,2-bis(olcoyloxy)-3-(trimethylammonio)-propaneand at least one cholesterol or cholesterol derivative forming heatedlipid components; ii) hydrating said heated lipid components forminghydrated lipid components; iii) sonicating said hydrated lipidcomponents forming sonicated lipid components; iv) extruding saidsonicated lipid components sequentially through filters of decreasingpore size; and v) adding a biologically active agent to said extrudedlipid components forming invaginated vase-like liposomes.
 2. Theliposome of claim 1, wherein the biologically active agent is DNA,thereby forming a DNA sandwich liposome.
 3. The liposome according toclaim 2 further comprising a targeting ligand to the exterior surface ofsaid DNA-sandwich liposome.
 4. The liposome according to claim 2 furthercomprising a second biologically active agent.
 5. The liposome of claim2 wherein the DNA, 1,2-bis(oleoyloxy)-3-(trimethylammonio)-propane andat least one cholesterol or cholesterol derivative carry a net chargevalue of
 2. 6. A DNA-sandwich composite liposome comprising aninvaginated vase-like structure having a plurality of lipid bilayers,and a DNA molecule positioned between two or more lipid bilayers of thesandwich liposome, having net charge of 2 and a size of 200-450 nm.
 7. Amethod for preparing invaginated vase-like liposomes comprising thesteps of: i) heating a mixture of1,2-bis(olcoyloxy)-3-(trimethylammonio)-propane and at least one ofcholesterol or cholesterol derivative forming heated lipid components;ii) hydrating said heated lipid components forming hydrated lipidcomponents; iii) sonicating said hydrated lipid components formingsonicated lipid components; iv) extruding said sonicated lipidcomponents sequentially through filters of decreasing pore size forminginvaginated vase-like liposomes; and v) adding DNA to said invaginatedvase-like liposomes forming DNA-sandwich liposomes.